The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. More than 300 abnormal hemoglobins have been discovered upon examination of patients with clinical symptoms and by electrophoretic surveys of a clinically normal population. Many of these abnormalities result in clinical pathologies having altered hemoglobin levels or hemoglobin having an altered ability to bind oxygen. Among these diseases are sickle cell anemia, both α and β-thalassemias and hemoglobin M.
An ability to measure hemoglobin (Hgb) in blood samples is an essential part of diagnostic analysis and is also important for monitoring responsiveness to therapies directed towards diseases which affect hemoglobin and to therapies directed towards other diseases, which may have adverse side effects on the hemoglobin level.
White blood cells in the peripheral blood of normal subjects consist of five types, i.e., lymphocytes, monocytes, neutrophils, eosinophils and basophils. The latter three types of white blood cells are collectively referred to as granulocytes. Counting and differentiating different types of white blood cells in a blood sample provides valuable information for clinical diagnosis.
The classification and counting of white blood cells has most commonly been conducted by the differential analysis method which is also referred to as the manual method. Automatic blood analyzers are also commonly used for counting white blood cells, employing a lytic reagent to lyse red blood cells and then measuring the remaining white blood cells. A more sophisticated apparatus has been developed that counts different types of white blood cells (differential analysis of) including monocytes, lymphocytes and granulocytes. Ideally, one would like to be able to accomplish multiple diagnostic analyses such as hemoglobin measurement and counting the numbers of white blood cells or differential analysis of white blood cell subpopulations in a single automated step.
Among the many well-known methods for hemoglobin determination, the cyanide hemoglobin method has been recommended as a standard by the International Committee for Standardization in Hematology. Modification of this method by Matsubara and Okuzono has led to its wide usage in clinical laboratories. In this method, the iron ion of the heme group in all forms of hemoglobin of the red cells is oxidized to methemoglobin by potassium ferricyanide. The methemoglobin is then complexed with the cyanide anion, which has a very high affinity to the iron ion of the heme group, and forms a cyanmethemoglobin chromogen. This extremely stable chromogen has a maximum absorption at 540 nm, which is measured manually by spectrophotometer.
Despite the stable chromogens formed by the standard cyanmethemoglobin method and its modified automatic methods, the reagent waste has caused enormous environmental concern because of the potassium cyanide used. In the last twenty years, a tremendous effort has been undertaken to develop automated hemoglobin analysis methods without utilizing cyanide.
Oshiro et al, Clin. Biochem. 1583 (1982), teach the use of a reagent for hemoglobin analysis that comprises sodium laurylsulfate (SLS) and Triton X-100 (a nonionic surfactant) in a neutral pH (7.2). The SLS is used to lyse red blood cells and is believed to further produce a SLS-hemoglobin complex which has a maximum absorption at 539 nm and a shoulder at 572 nm. The reaction completes within 5-10 minutes and the total hemoglobin measurement is quantitative. However, as later taught in U.S. Pat. No. 5,242,832 (to Sakata), it is not possible with Oshiro's method to analyze white blood cells simultaneously with hemoglobin measurement.
U.S. Pat. No. 5,242,832 (to Sakata) discloses a cyanide-free lysing reagent for counting white blood cells and measuring the hemoglobin concentration in blood samples. The lysing reagent comprises at least one first surfactant which is a quaternary ammonium salt, at least one second surfactant which includes cationic and amphoteric surfactants, and at least one hemoglobin stabilizer selected from the group including Tiron, 8-hydroxyquinoline, bipyridine, 1-10-phenanthroline, phenolic compounds, bisphenol, pyrazole and derivatives, second phenyl 5-pyrazolone and derivatives, phenyl 3-pyrazolone, and imidazole and its derivatives. Sakata teaches that fractionation of the white blood cells into two or three groups including an aggregate of lymphocytes, an aggregate of monocytes, eosinophils and basophils, and an aggregate of neutrophils can only be accomplished by using at least two suitable surfactants and by rigorously controlling the surfactant concentration. Sakata also teaches that the preferred pH range of the lysing reagent is from 5.0 to 8.0. If the pH value is 3.0 or less, damage to the white blood cells increases thus rendering measurement of white blood cells difficult, and if the pH is 9.0 or more, the stability of hemoglobin deteriorates over time.
PCT/US95/02897 (Kim) discloses a cyanide-free reagent for determining hemoglobin in a whole blood sample. The reagent comprises a ligand selected from the group consisting of imidazole and derivatives, N-hydroxyacetamide, H-hydroxylamine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline and isoquinoline, and a surfactant with a strong erythrolytic capability selected from the group consisting of lauryl dimethylamine oxide and octylphenoxy polyethoxyethanol. The analysis method is fast, less than 10 seconds. However, the reagent only performs under an extreme alkaline condition, pH from 11 to 14. In addition, no capability of counting white blood cells or differentiating white blood cell subpopulations is taught by Kim.
U.S. Pat. Nos. 5,763,280 and 5,882,934 (to Li et al.) disclose cyanide-free reagents containing an organic ligand for measurement of hemoglobin and white blood cells in a blood sample. The organic ligand can be triazole and its derivatives, tetrazole and its derivatives, alkaline metal salts of oxonic acid, melamine, aniline-2-sulfonic acid, quinaldic acid, 2-amino-1,3,4-thiadiazole, triazine and its derivatives, urazole, DL-pipecolinic acid. isonicotinamide, anthranilonitrile, 6-aza-2-thiothymine, adenine, 3-(2-thienyl)acrylic acid, benzoic acid, alkali metal or ammonium salt of benzoic acid, or pyrazine and its derivatives.
U.S. Pat. No. 6,740,527 (to Wong et al.) discloses cyanide-free reagents for measurement of hemoglobin and white blood cells in a blood sample. The reagents contain at least one hydroxylamine salt selected from the group consisting of hydrochloride, sulfate, phosphate, and other acid salts.
A need is still present for a cyanide-free lytic reagent that contains a ligand that is inexpensive and environmentally friendly for a relatively large consumption on routine hematology analyzers.